scholarly journals TRAP1 Regulates Wnt/β-Catenin Pathway through LRP5/6 Receptors Expression Modulation

2020 ◽  
Vol 21 (20) ◽  
pp. 7526
Author(s):  
Giacomo Lettini ◽  
Valentina Condelli ◽  
Michele Pietrafesa ◽  
Fabiana Crispo ◽  
Pietro Zoppoli ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cellular Differentiation ◽  
Hct116 Cell ◽  
Reporter Assay ◽  
Colorectal Carcinomas ◽  
Cancer Stemness ◽  
Protein Ubiquitination ◽  
Ligand Receptors ◽  
Multiple Levels

Wnt/β-Catenin signaling is involved in embryonic development, regeneration, and cellular differentiation and is responsible for cancer stemness maintenance. The HSP90 molecular chaperone TRAP1 is upregulated in 60–70% of human colorectal carcinomas (CRCs) and favors stem cells maintenance, modulating the Wnt/β-Catenin pathway and preventing β-Catenin phosphorylation/degradation. The role of TRAP1 in the regulation of Wnt/β-Catenin signaling was further investigated in human CRC cell lines, patient-derived spheroids, and CRC specimens. TRAP1 relevance in the activation of Wnt/β-Catenin signaling was highlighted by a TCF/LEF Cignal Reporter Assay in Wnt-off HEK293T and CRC HCT116 cell lines. Of note, this regulation occurs through the modulation of Wnt ligand receptors LRP5 and LRP6 that are both downregulated in TRAP1-silenced cell lines. However, while LRP5 mRNA is significantly downregulated upon TRAP1 silencing, LRP6 mRNA is unchanged, suggesting independent mechanisms of regulation by TRAP1. Indeed, LRP5 is regulated upon promoter methylation in CRC cell lines and human CRCs, whereas LRP6 is controlled at post-translational level by protein ubiquitination/degradation. Consistently, human CRCs with high TRAP1 expression are characterized by the co-upregulation of active β-Catenin, LRP5 and LRP6. Altogether, these data suggest that Wnt/β-Catenin signaling is modulated at multiple levels by TRAP1.

scholarly journals Deficient or R273H and R248W Mutations of p53 Promote Chemoresistance to 5-FU via TCF21/CD44 Axis-Mediated Enhanced Stemness in Colorectal Carcinoma

2022 ◽  
Vol 9 ◽  
Author(s):  
Xiaolei Gao ◽  
Xuan Zheng ◽  
Yixin Zhang ◽  
Liying Dong ◽  
Liangjie Sun ◽  
...  
Keyword(s):  
Colorectal Carcinoma ◽  
Cancer Cells ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Hct116 Cell ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Mutant P53 ◽  
Reporter Assay ◽  
Wild Type

Background: p53 mutations are highly frequent in various human cancers and are reported to contribute to tumor malignance and chemoresistance. In this study, we explored the mechanism by which mutant p53 promotes carcinogenesis and chemoresistance and provided novel insights into cancer therapy.Materials and methods: A total of 409 patients with colorectal carcinoma from TCGA database were subdivided into two groups according to the p53 status, namely, mutant p53 and wild-type p53, following with GSEA analysis. The differences of the clinicopathologic index were also analyzed. Two HCT116 cell lines containing hot spots at codons R273H and R248W of p53 were constructed based on HCT116 with knockout p53, respectively. Cell viability, mobility, clonogenesis, and stemness were detected by CCK8, transwell migration and invasion, colonogenic, and sphere formation assays. Resistance to 5-FU was examined by live-dead staining and flow cytometry. qPCR, Western blot, and luciferase reporter assay were performed to identify that deficient or mutant p53 promoted chemoresistance of the colorectal carcinoma cell line HCT116 through the TCF21/CD44 signaling pathway, with the following rescue assays by overexpression of TCF21 and knockdown of CD44.Results: Patients with recurrence harbor a higher frequency of mutant p53 than those without recurrence (p < 0.05). The mutant p53 group developed a larger tumor than the wild-type one. GSEA analysis showed that oncogenic signatures were enriched in the mutant p53 group. Extracellular assays showed that cancer cells with deficient or mutant p53 (R273H and R248W, respectively) promoted colon cancer cell growth, migration, invasion, and stemness. The mutant cancer cells were also observed to be significantly resistant to 5-FU. Xenografts also confirmed that HCT116 cells harboring deficient or mutant p53 promoted cancer growth and 5-FU tolerance. Luciferase reporter assay showed that deficient or mutant p53 R237H and R248W endowed cancer cells with chemoresistance by activating CD44 via repressing the nuclear transcription factor TCF21 expression. Overexpression of TCF21 or knockdown of CD44 could rescue the sensitivity to 5-FU in deficient and mutant p53 HCT116 cell lines.Conclusion: Our results, for the first time, reveal a novel deficient or mutant p53/TCF21/CD44 signaling pathway which promotes chemoresistance in colorectal carcinoma. The axis could be an effective therapeutic strategy against deficient- or mutant p53-driven chemoresistance.

scholarly journals MiR-624-5p enhances cell resistance against cisplatin via PDGFRA/Stat3/PI3K axis in ovarian cancer

2020 ◽  
Vol 19 (4) ◽  
pp. 691-698
Author(s):  
Lin I-Ju ◽  
Tian YongJie
Keyword(s):  
Ovarian Cancer ◽  
Cell Viability ◽  
Cell Line ◽  
Cell Lines ◽  
Clinical Stage ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay

Purpose: The purpose of this study was to evaluate the role of miR-624-5p in ovarian cancer.Methods: MiR-624-5p expression in ovarian cancer {OC) cell lines and normal cells (NCs) was evaluated and compared the differential miR-624-5p in OC A2780 cells and cisplatin-resistant OC cell line (A2780/DDP). CCK-8 was used to evaluate changes in cell viability of the A2780 and A2780/DDP cell lines as well as silenced miR-624-5p. Western Blot examined the Stat3 and phosphorylated Pi3k. The binding between PDGFRA and miR-624-5p was predicted on Targetscan and verified through Luciferase Reporter Assay. The role of PDGFRA in A2780/DDP by overexpressing PDGFRA was evaluated by RT-qPCR and CCK-8 assays. RT-qPCR assay also measured miR-624-5p expression responsive to different dosages of cisplatin and CCK8 examined viability levels correspondingly. In addition, the interplay of PDGFRA and miR-624-5p by combined downregulation of both miR-624-5pand PDGFRA were evaluated.Results: OC cells had higher miR-624-5p expression than NCs but lower compared to cisplatinresistant A2780/DDP cells. A2780/DDP cells had higher viability than OC cell line A2780. Stat3 and phosphorylated PI3K were activated in A2780/DDP cells. Silencing miR-624-5p led to lower viability inA2780/DDP cells. miR-624-5p expression dropped as the cisplatin concentration increased, resulting in decreasing viability respectively. Luciferase Reporter assay validated the binding of miR-624-5p and PDGFRA in A2780/DDP cells. Overexpressed PDGFRA induced lower cell viability in A2780/DDP cells. Downregulation of PDGFRA partially restored the lowered viability and inhibited Stat3 as well as phosphorylated Pi3k induced by miR-624-5p inhibitor.Conclusion: MiR-624-5p could add to the cellular resistance to cisplatin in OC in-vitro model, which indicated that it might help unveil the mystery of drug-resistance in clinical stage of ovarian cancer. Keywords: MiR-624-5p, resistance, cisplatin, PDGFRA/Stat3/PI3K, ovarian cancer

scholarly journals MicroRNA-103 Promotes Proliferation and Inhibits Apoptosis in Spinal Osteosarcoma Cells by Targeting p57

2018 ◽  
Vol 26 (6) ◽  
pp. 933-940 ◽  
Author(s):  
Xuesong Wang ◽  
Yong Lin ◽  
Lei Peng ◽  
Ruifu Sun ◽  
Xiaojin Gong ◽  
...  
Keyword(s):  
Cell Survival ◽  
Cell Lines ◽  
Poor Prognosis ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Osteosarcoma Cells ◽  
Binding Effect ◽  
Mg63 Cells

Osteosarcoma is one of the most aggressive malignancies with poor prognosis rates. Many studies have demonstrated that miRNAs were involved in osteosarcoma, but the role of miR-103a in osteosarcoma remains elusive. In this study, we detected the expression levels of miR-103 in osteosarcoma and non-osteosarcoma tissues and cell lines. The binding effect of miR-103 on p57 was detected by luciferase reporter assay. After altering expressions of miR-103 or p57, viability, migration, invasion, and apoptosis of MG63 cells and expressions of proteins related with the JNK/STAT and mTOR pathways were all detected. We found the higher expression of miR-103 in osteosarcoma tissues and cell lines compared with non-osteosarcoma tissues and cell lines. miR-103 overexpression promoted survival, migration, and invasion of MG63 cells. Knockdown of miR-103a inhibited cell survival, migration, and invasion by upregulating the expression of p57, which was a target of miR-103. Moreover, miR-103a overexpression activated the JNK/STAT and mTOR pathways probably through inhibiting p57 expression. In conclusion, miR-103a acted as an oncogene in osteosarcoma, probably through activating the JNK/STAT and mTOR pathways by inhibiting p57 expression.

scholarly journals Mcl-1 Is a Novel Target of miR-26b That Is Associated with the Apoptosis Induced by TRAIL in HCC Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Chunlin Jiang ◽  
Jianting Long ◽  
Baoxian Liu ◽  
Xiaoyan Xie ◽  
Ming Kuang
Keyword(s):  
Cell Lines ◽  
Bioinformatic Analysis ◽  
Negative Regulator ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Mrna Transfection ◽  
Novel Target ◽  
Hcc Cells

Aim. To investigate the role of miR-26b and Mcl-1 in TRAIL-inducing cell death in hepatocellular carcinoma.Methods. The expression of miR-26b and Mcl-1 in HCC was detected by RT-qPCR and western blot. The regulation of Mcl-1 by miR-26b was determined by luciferase reporter assay. MTT and flow cytometry were employed to detect the cell viability and apoptosis.Results. miR-26b is commonly downregulated in HCC cell lines compared with the LO2 cell line. In contrast, the Mcl-1 expression is upregulated in HCC cell lines. Bioinformatic analysis identified a putative target site in the Mcl-1 mRNA for miR-26b and luciferase reporter assay showed that miR-26b directly targeted the 3′-UTR (3′-Untranslated Regions) of Mcl-1 mRNA. Transfection of miR-26b mimics suppressed Mcl-1 expression in HCC cells and sensitized the cancer cells to TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) cytotoxicity. In addition, transfection of HCC cells with Mcl-1 expression plasmid abolished the sensitization effect of miR-26b to TRAIL-inducing apoptosis.Conclusions. Our study showed that miR-26b was a negative regulator of Mcl-1 gene and sensitized TRAIL-inducing apoptosis in HCC cells, suggesting that the miR-26b-Mcl-1 pathway might be a novel target for the treatment of HCC.

scholarly journals MicroRNA-221 Targets FBXW11 to Inhibit NPC Cell Proliferation by Regulating PTEN Signaling

2021 ◽  
Author(s):  
Jun Liu ◽  
Chengyi Song ◽  
Zhuoping Liang ◽  
Xiang Long ◽  
Min Guo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Biological Function ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Cell Cycle Transition ◽  
Polymerase Chain ◽  
Molecular Regulatory Mechanisms

Abstract Background: In the current study, we aim to demonstrate the biological function and molecular regulatory mechanisms of miR-221 in human nasopharyngeal carcinoma (NPC). Methods: The quantitative real time-polymerase chain reaction was used to measure the expression of miR-221 in NPC clinical tissues and cells. And the flow cytometry assay was used to demonstrate the role of miR-221 on cell cycle, and the potential target of miR-221 was predicted and identified using luciferase reporter assay.Results: Our results demonstrate that miR-221 expression was significantly decreased in NPC tissues and cell lines. We also confirmed that inhibition of miR-221 could induce G1/S cell cycle transition through upregulation of the cyclin D-CDK4/6 complex but not cyclin E-CDK2 complexes. Furthermore, luciferase reporter assay demonstrated that miR-221 could directly bind to the 3’-UTR of FBXW11. FBXW11 expression was found to increase in NPC, and was inversely correlated with miR-221 expression; thus, FBXW11 expression interfered in the biological function of miR-221. We further confirmed that miR-221 targeted FBXW11 to inhibit proliferation and promote apoptosis in NPC cell lines through regulating the PTEN signaling pathway. Conclusion: our findings suggest that miR-221 plays an important role as a tumor suppressive factor in the occurrence and progression of NPC.

scholarly journals Potential Onco-Suppressive Role of miR122 and miR144 in Uveal Melanoma through ADAM10 and C-Met Inhibition

Cancers ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1468 ◽  
Author(s):  
Adriana Amaro ◽  
Michela Croce ◽  
Silvano Ferrini ◽  
Gaia Barisione ◽  
Marina Gualco ◽  
...  
Keyword(s):  
Cell Lines ◽  
Uveal Melanoma ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Primary Tumors ◽  
Cancer Genome Atlas ◽  
Data Portal ◽  
Low Levels ◽  
Tcga Dataset

Uveal melanoma (UM) is a rare tumor of the eye that leads to deadly metastases in about half of the patients. ADAM10 correlates with c-Met expression in UM and high levels of both molecules are related to the development of metastases. MiR122 and miR144 modulate ADAM10 and c-Met expression in different settings. We hypothesized a potential onco-suppressive role for miR122 and miR144 through modulation of ADAM10 and c-Met in UM. We analyzed the UM Cancer Genome Atlas data portal (TCGA) dataset, two other cohorts of primary tumors and five human UM cell lines for miR122 and miR144 expression by miR microarray, RT-qPCR, Western blotting, miR transfection and luciferase reporter assay. Our results indicate that miR122 and miR144 are expressed at low levels in the UM cell lines and in the TCGA UM dataset and were down-modulated in a cohort of seven UM samples, compared to normal choroid. Both miR122 and miR144 directly targeted ADAM10 and c-Met. Overexpression of miR122 and miR144 led to reduced expression of ADAM10 and c-Met in the UM cell lines and impaired cell proliferation, migration, cell cycle and shedding of c-Met ecto-domain. Our results show that miR122 and miR144 display an onco-suppressive role in UM through ADAM10 and c-Met modulation.

scholarly journals FOXO4 Inhibits the Migration and Metastasis of Colorectal Cancer by Regulating the APC2/β-Catenin Axis

2021 ◽  
Vol 9 ◽  
Author(s):  
Yan Sun ◽  
Lin Wang ◽  
Xuehu Xu ◽  
Puqing Han ◽  
Jinghao Wu ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Epithelial Mesenchymal Transition ◽  
Hct116 Cell ◽  
Suppressor Gene ◽  
Mesenchymal Transition ◽  
Normal Tissues ◽  
Forkhead Box ◽  
The Relationship

Objective: Adenomatous polyposis coli 2 (APC2) is a colorectal cancer (CRC) tumor-suppressor gene. The progression of several kinds of cancer is closely associated with Forkhead box O4 (FOXO4). However, the function of FOXO4 in CRC is unclear. This study focused on the role of FOXO4 and the relationship between FOXO4 and APC2 in CRC migration and metastasis.Methods: The expressions of FOXO4, APC2, and p(S37)-β-catenin were detected in CRC tissues by immunohistochemistry, and their correlation was analyzed using the Spearman coefficient. Chromatin immunoprecipitation was used to test whether FOXO4 binds and regulates APC2 as a transcription factor. Either FOXO4 overexpression or APC2 knockdown was performed in CRC cell lines. The roles of FOXO4 and APC2 were investigated in CRC migration and metastasis.Results: FOXO4 was downregulated in CRC tissues compared with normal tissues and positively correlated with APC2 and p(S37)-β-catenin. FOXO4 could combine the promoter region of APC2 to upregulate its expression and increase the phosphorylated degradation of β-catenin. Stemness genes (CD133, ABCG1, and SOX2) were inhibited by FOXO4 overexpression in SW620 and HCT116 cell lines. Overexpressed FOXO4 suppressed epithelial–mesenchymal transition and the migration of CRC cell lines and metastasis of HCT116 in both the spleen and liver of nude mice, which was reversed by APC2 knockdown.Conclusion: This research demonstrates that overexpressed FOXO4 inhibits the migration and metastasis of CRC cells by enhancing the APC2/β-catenin axis, suggesting that FOXO4 is a potential therapeutic target of CRC.

scholarly journals miR-651-3p Enhances the Sensitivity of Hepatocellular Carcinoma to Cisplatin via Targeting ATG3-Mediated Cell Autophagy

2021 ◽  
Vol 2021 ◽  
pp. 1-7
Author(s):  
Lei Zou ◽  
Peng Sun ◽  
Lei Zhang
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Lines ◽  
Luciferase Reporter ◽  
Chemotherapeutic Drugs ◽  
Reporter Assay ◽  
Flow Cytometry Assay ◽  
Downstream Target ◽  
Autophagy Pathway ◽  
Dual Luciferase Reporter Assay

Drug resistance is a major challenge for hepatocellular carcinoma (HCC) treatment in a clinic, which limits the therapeutic effect of the chemotherapeutic drugs, including cisplatin (CDDP), in this disease. Mounting evidence has identified that miRNAs dysfunction is related to the resistance of tumor cells to CDDP, and miR-651-3p has been identified as a tumor inhibitor to suppress the progression of multiple tumors. However, the role of miR-651-3p in HCC remains unclear. In this study, the relative expression of miR-651-3p in HCC tissues and cell lines were measured, and the functions of miR-651-3p were also observed by CCK-8 assay, flow cytometry assay, and Western blot. Moreover, the downstream target of miR-651-3p was predicted and verified via TargetScan and dual-luciferase reporter assay, and its functions were also investigated. The results showed that miR-651-3p was significantly downregulated in HCC tissues and cell lines, and the decreased miR-651-3p was also observed in CDDP-induced cells. miR-651-3p upregulation could effectively inhibit the proliferation and induce the apoptosis of R-HepG2. It was also found that ATG3 was a downstream target of miR-651-3p, and ATG3 was highly upregulated in HCC tissues. Moreover, the upregulated ATG3 could partly reverse the effects of miR-651-3p on R-HepG2. Besides, miR-651-3p involved the autophagy pathway of the HCC cells via targeting ATG3. In conclusion, miR-651-3p could regulate the autophagy to enhance the sensitivity of HepG2 cells to CDDP via targeting ATG3.

Glioblastoma invasion and NMDA receptors: A novel prospect

2019 ◽  
Vol 106 (3) ◽  
pp. 250-260 ◽  
Author(s):  
DN Nandakumar ◽  
P Ramaswamy ◽  
C Prasad ◽  
D Srinivas ◽  
K Goswami
Keyword(s):  
Glutamate Receptors ◽  
Cell Lines ◽  
Intracellular Signaling ◽  
Transcript Level ◽  
Glioblastoma Cells ◽  
Invasion And Migration ◽  
Matrigel Invasion ◽  
Nmdar Activation ◽  
And Migration

Purpose Glioblastoma cells create glutamate-rich tumor microenvironment, which initiates activation of ion channels and modulates downstream intracellular signaling. N-methyl-D-aspartate receptors (NMDARs; a type of glutamate receptors) have a high affinity for glutamate. The role of NMDAR activation on invasion of glioblastoma cells and the crosstalk with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is yet to be explored. Main methods LN18, U251MG, and patient-derived glioblastoma cells were stimulated with NMDA to activate NMDAR glutamate receptors. The role of NMDAR activation on invasion and migration and its crosstalk with AMPAR were evaluated. Invasion and migration of glioblastoma cells were investigated by in vitro trans-well Matrigel invasion and trans-well migration assays, respectively. Expression of NMDARs and AMPARs at transcript level was evaluated by quantitative real-time polymerase chain reaction. Results We determined that NMDA stimulation leads to enhanced invasion in LN18, U251MG, and patient-derived glioblastoma cells, whereas inhibition of NMDAR using MK-801, a non-competitive antagonist of the NMDAR, significantly decreased the invasive capacity. Concordant with these findings, migration was significantly augmented by NMDAR in both cell lines. Furthermore, NMDA stimulation upregulated the expression of GluN2 and GluA1 subunits at the transcript level. Conclusions This study demonstrated the previously unexplored role of NMDAR in invasion of glioblastoma cells. Furthermore, the expression of the GluN2 subunit of NMDAR and the differential overexpression of the GluA1 subunit of AMPAR in both cell lines provide a plausible rationale of crosstalk between these calcium-permeable subunits in the glutamate-rich microenvironment of glioblastoma.

Colorectal carcinomas - Role of p53

2019 ◽  
Vol 11 (3) ◽  
pp. 144-149
Author(s):  
Sindu V ◽  
◽  
Priyadharshini M ◽  
Keyword(s):  
Colorectal Carcinomas
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